INVESTIGATION

Homogeneous Nuclear Background for Mitochondrial Cline in Northern Range of Notochthamalus scabrosus

Christina Zakas,* Ken Jones,† and John P. Wares‡,1 *New York University, Center for Genomics and Systems Biology, New York, New York 10003, †University of Colorado, Aurora, Colorado 80045, and ‡University of Georgia, Athens, Georgia 30602

ABSTRACT A mitochondrial cline along the Chilean coast in the Notochthamalus scabrosus KEYWORDS suggests a species history of transient allopatry and secondary contact. However, previous studies of RAD-seq nuclear sequence divergence suggested population genetic homogeneity across northern and central SNP Chile. Here, we collect single-nucleotide polymorphism data from pooled population samples sequenced cytonuclear with restriction site2associated DNA sequencing procedures, confirm these data with the use of a Golden- disequilibrium Gate array, and identify a discordance between population genetic patterns in the nuclear and mitochon- barnacle drial genomes. This discordance was noted in previous work on this species, but here it is confirmed that the Chile nuclear genome exhibits only slight phylogeographic variation across 3000 km of coastline, in the presence of a strong and statistically significant mitochondrial cline. There are nevertheless markers (approximately 5% of nuclear single-nucleotide polymorphisms) exhibiting cytonuclear disequilibrium relative to mitotype. Although these data confirm our previous explorations of this species, it is likely that some of the nuclear genomic diversity of this species has yet to be explored, as comparison with other barnacle phylogeography studies suggest that a divergence of similar magnitude should be found in the nuclear genome somewhere else in the species range.

Patterns of mitochondrial diversity have heralded great discoveries in Pavlova et al. 2013). For example, divergence of mitochondrial lin- biodiversity and often are used to indicate the likelihood that evolu- eages in an Australian robin (Eopsaltria australis) exhibits an east- tionarily distinct lineages are harbored within recognized morpholog- west pattern, perpendicular to the latitudinal structure of nuclear ical species (Hebert et al. 2004; Moritz and Cicero 2004). However, markers (Pavlova et al. 2013). divergence of mitochondrial lineages is not necessarily representative of Another such case of discordance has been identified in the the rest of a eukaryotic genome (Hudson and Coyne 2002; Hickerson barnacle Notochthamalus scabrosus (Foster and Newman 1987) along et al. 2006). In some cases, exploration of divergence at nuclear the central coast of Chile (Zakas et al. 2009). Two divergent mito- markers supports a general pattern (Tellier et al. 2009), but in other chondrial lineages are found in this species, with one of them only cases the two genomic compartments appear to have wildly distinct found in natural populations south of ~30°S(Zakaset al. 2009; Laughlin histories of isolation and/or selection (Toews and Brelsford 2012; et al. 2012). A nuclear gene (elongation factor 1-a; nEF1) surveyed for the same region showed no such geographic pattern of genetic fi Copyright © 2014 Zakas et al. diversi cation (Zakas et al. 2009), despite the capacity for this gene doi: 10.1534/g3.113.008383 region to mirror such patterns and magnitudes of divergence in Manuscript received September 9, 2013; accepted for publication November 25, other coastal (Sotka et al. 2004). Because the nEF1 data 2013; published Early Online December 17, 2013. did not exhibit the expected genealogical divergence, as seen in other This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License (http://creativecommons.org/licenses/ barnacle species with similar mitochondrial clines (Sotka et al. by/3.0/), which permits unrestricted use, distribution, and reproduction in any 2004), it is clear that a better representation of diversity and diver- medium, provided the original work is properly cited. gence in the nuclear genome will be needed to understand the Supporting information is available online at http://www.g3journal.org/lookup/ phylogeography of N. scabrosus. suppl/doi:10.1534/g3.113.008383/-/DC1. Such a result suggests a discordant history for the mitochondrial Data from this article are available NCBI BioProject under PRJNA227359. 1Corresponding author: Department of Genetics, University of Georgia, Athens, GA and nuclear genomes of N. scabrosus, but of course one nuclear locus 30602. E-mail: [email protected] is not sufficiently representative of the dynamics of the genome.

Volume 4 | February 2014 | 225 Recent advances in DNA sequencing technology (Schuster 2008) for individuals comprised solely of the A1, A2 (“northern”), and B make it readily possible to efficiently survey a larger fraction of the (“southern”) mitochondrial haplotypes as described in Laughlin genome for population genetic analysis. Elucidating the pattern of et al. (2012). population divergence by the use of many thousands of nuclear loci Pooled genomic DNA were cut per standard protocols (Peterson reveals the potential combinations of ecological and evolutionary et al. 2012) by the use of SpeI and Sau3AI, chosen for their utility in forces that drive this mismatch in genome histories. Here we use similar projects (K. Jones, unpublished results). Libraries were then a combination of reduced-representation genome sequencing (restric- prepared for Illumina sequencing by the use of TruSeq adapters and tion site2associated DNA sequencing, or RADseq; Peterson et al. 100-bp paired ends were sequenced on an Illumina HiSequation 2500 2012) of population samples and direct multilocus genotyping of at the University of Colorado at Denver core facility. markers developed from such data to find nuclear regions that may be associated with the divergent mitochondrial lineages found in Genome-wide single-nucleotide polymorphism N. scabrosus. Because the aforementioned nEF1 data did not exhibit (SNP) analysis a cline, we were interested in determining the extent to which a large RADseq data were trimmed to remove adapter sequence and error- representation of the nuclear genome displayed a concordant pattern. prone regions. Sequence fragments were aligned and organized with We also assess the utility of pooled RADseq data by comparison with the program STACKS (Catchen et al. 2013). Because these data rep- direct genotyping of individuals in a subset of populations; the op- resent pooled samples from distinct locations, the consensus stacks portunity to collect appropriate data via pooled sampling, if it provides were used as the reference library in the program POPOOLATION2 (Kofler fi results consistent with individual genotypes, could be an ef cient and et al. 2011), which aligns reads from pooled libraries to these reference inexpensive way to identify genome-wide diversity in nonmodel sequences and generates allele frequency and other population genetic organisms. statistics such as FST. SNPs were only included for analysis with a minimum fragment coverage of 50 and minimum quality score of 20. METHODS Specimen collection and sequencing SNP selection Collected specimens and DNA isolation protocols are described in To genotype at the individual level, SNPs were chosen for assay using detail in Laughlin et al. (2012). Sample locations are shown in Figure 1. the Illumina BeadXPress platform as in Zakas and Wares (2012). Pooled samples of each location, with separate pools for “low” and Thus, SNPs were identified that had sequenced flanking regions of “high” intertidal samples from Temblador and Guanaqueros, were .30 bp for primer design; these were scored by Illumina for their generated for each site with equimolar concentrations of each in- probability of success. Only SNPs with the greatest possible quality dividual. All individuals included had been previously sequenced at ranking were selected for further analysis. We chose 192 for multi- mitochondrial cytochrome oxidase I (COI) and identified to mito- locus genotyping, setting specific aprioricriteria for selection follow- chondrial type. Three separate genomic DNA pools were generated ing Zakas et al. (2012), where ribosomal markers were eliminated and a balance of SNPs from coding regions and anonymous regions, as well as apparent nonsynonymous polymorphisms and synonymous polymorphisms were chosen. Consensus sequences for each region were evaluated with NCBI BLAST; any contig that blasted to a mito- chondrial region with score e , 0.001 was considered a likely mito- chondrial region and was excluded. We further screened the SNP library for potential outliers that exhibit high FST values among locations using the program BAYESSCAN (Foll and Gaggiotti 2008); these outliers were included in the BeadX- Press assay. To minimize potential ascertainment biases in selection of SNPs for completion of the array, SNPs with minor allele frequencies of 0.3020.35, 0.3620.40, 0.4120.45, and 0.4620.50 were equally represented. Markers with lower minor allele frequencies were elim- inated to guard against sequencing artifacts as in Zakas et al. (2012). Further, only one SNP was selected from each identified sequence “stack” (contig) for the BeadXPress array to minimize linkage disequi- librium among markers.

BeadXPress assay data Genomic DNA samples at 20250 ng/mL for individuals from three representative locations (Arica, Las Cruces, and Niebla; Figure 1) were prepared by the Georgia Genomics Facility and run on a BeadXPress platform (Illumina). Resultant data were analyzed and clustered as in Zakas and Wares (2012), with a minimum genotype call score of 0.30. Figure 1 Locations of Notochthamalus scabrosus collections used in More than 10% of individual DNA samples were genotyped a second RADseq and SNP analysis (sample sizes indicate input for one or more time to determine genotype error rates (Pompanon et al. 2005) using libraries from each location for RADseq). All locations were included in an R script written by J.P.W. Loci with a failure rate greater than 30% RADseq analysis; sites marked in yellow were used for GoldenGate were excluded from further analysis. Data were exported and basic SNP analysis. analyses performed using GenAlEx 6.5 (Peakall and Smouse 2006).

226 | C. Zakas, K. Jones, and J. P. Wares Population structure per population of 3.62 6 1.21 million in each direction. Data may be Multilocus genotype data for the representative populations were accessed from the Short Read Archive at NCBI (BioProject evaluated for patterns of allelic diversity and heterozygosity, including PRJNA227359). standard F-statistics, to identify loci that may violate Hardy-Weinberg assumptions. Multilocus G statistics, including corrected G’st (Hedrick Genome-wide SNP analysis 2005), were calculated across sampled locations using GENALEX 6.5 We identified informative SNP loci in two ways: first we analyzed (Peakall and Smouse 2006) with significance assessed through 1000 SNPs based on groupings by mitochondrial clade (A1, A2, and B as permutations. identified in Laughlin et al. 2012). When these three mitochondrial Data were exported for analysis using STRUCTURE 2.3.4 (Pritchard populations were compared, POPOOLATION2 identified 58,605 biallelic et al. 2000), with 10 replicate analyses of 50,000 steps with 5000 steps SNPs that passed our threshold. Using an identical method across burn-in using sample location as prior and allowing admixture. Like- nine geographic populations, we identified 37,046 biallelic SNPs. A lihoods were recorded and interpreted using STRUCTUREHARVESTER subset of SNPs was not scored for one or more locations given the (Earl and Vonholdt 2012) to identify the most likely number of aforementioned criteria. One population in particular (high tidal evolutionary populations using the Evanno et al. (2005) criterion. range sample from Guanaqueros) was excluded based on poor re- covery of data in this sample. The pairwise calculations of FST between Cytonuclear disequilibrium (CND) Antofagasta, Las Cruces, and Niebla are shown in Figure 2 (average Data from the largest single population identified using STRUCTURE FST 0.012). Results are similar for all other pairwise population com- 6 were analyzed relative to mitochondrial type (A, “northern” or B, parisons, with average FST of 0.0108 0.002. Results are not shown “southern”)usingCNDD (statgen.ncsu.edu/cnd), which implements for the A1/A2/B pooled populations as our overall results indicate that the methods of Asmussen and Basten (1994) for calculating CND grouping individuals by mitotype has no effect on evolutionary di- in diallelic loci (the statistical association between mitochondrial hap- vergence that is distinguishable from spatial population structure. lotype and the particular alleles or genotypes of an individual). Exact probabilities were calculated for all test statistics, and instances where SNP selection A , fi allelic disequilibrium D M exhibited P 0.05 were identi ed. Of all SNPs reported, 12,339 had sufficient flanking sequence (minimum 30 nt on either side of variable site) for primer design RESULTS and scoring. A total of 11,358 SNPs had optimal quality rankings using the Illumina scoring criteria. BAYESCAN identified 43 SNPs that Specimen collection and sequencing had a probability 97% or greater of being a statistical outlier; in all Libraries were demultiplexed with custom python scripts at UCD cases these were high- FST outliers. A total of 438 SNPs had a minor before analysis, and barcode and adapter sequence was trimmed allele frequency of at least 30% across the mitochondrial A and B before subsequent analysis. A total of 94,161,790 reads across all lineage libraries. Of these, 192 were selected based on the criteria listed libraries were recovered for analysis, with an average number of reads above for the BeadXPress array (Supporting Information, Table S1).

Figure 2 Log-distributed FST values by SNP comparing pop- ulations Arica (Ari), Las Cruces (LCr), Niebla (Nie; see Figure 1). Box plots indicate median

FST for all data sets of ,0.02. For each pairwise comparison, plot is shown for SNPs recov- ered through pooled population genomic calculation across 37,047 SNPs as well as the 102 filtered SNPs scored using BeadXPress arrays (indicated “bxp102”).

Volume 4 February 2014 | Low SNP Variation in Notochthamalus | 227 BeadXPress assay data n Table 1 Loci exhibiting significant cytonuclear disequilibrium A P After the removal of duplicate individual genotypes and identifying between allele and mitotype (D M statistic and value) or P loci that met quality criteria (see Materials and Methods), sufficient genotype and mitotype (statistics and values shown for AA, Aa, and aa genotype arbitrarily assigned to each diallelic locus) data were obtained for 102 biallelic loci in 147 individuals (Arica, n = A AA Aa aa 37; Las Cruces, n = 95; Niebla, n = 15). Only a single individual was Locus D M D M D M D M removed from analysis after inconsistency in genotype across two 32209_8940 6.205 (0.015) 0.317 (0.501) 6.675 (0.016) 7.354 (0.010) replicates; remaining data from 22 individuals with duplicate geno- 16301_4116 5.230 (0.012) 3.352 (0.105) 0.355 (0.593) 4.303 (0.062) 74853_9172 4.675 (0.038) 2.942 (0.132) 0.225 (0.792) 3.189 (0.097) types (14.9% of samples) indicated a low rate of allelic error (ea = 0.007). On average, 86.67% of loci were polymorphic in these samples, 23232_3413 2.994 (0.030) 8.363 (0.012) 0.363 (0.583) 1.498 (0.271) 111068_5411 4.183 (0.039) 5.043 (0.047) 1.733 (0.289) 1.026 (0.353) with an average of 1.87 alleles per locus. Observed heterozygosity (Ho = 0.286 6 0.009) was lower than expected heterozygosity (He = 0.328 6 0.008), suggesting a tendency for allelic dropout, paralogy, and/or population structure. Thirty-two loci exhibited FIS in excess of 0.2; Interestingly, this suggests that while the mitochondrial cline centers ° average FIS across all loci was 0.126. Individual heterozygosity (fre- at ~30 S, the mechanism for the strong mitochondrial divergence quency of heterozygous loci) ranged from 30.7% (Las Cruces) to likely originates in regions further to the south (J. Wares, unpublished 35.1% (Niebla). Two loci (780_10879 and 63806_7593) exhibit pri- data). The mitochondrial cline documented so far (Laughlin et al. vate alleles in Arica at low frequency (,0.01 across all samples), and 2012) appears to be somewhat independent of the nuclear background three loci (14707_1786, 8887_5638, and 30199_7401) exhibited pri- found in northern and central Chile. vate alleles in the Las Cruces sample, with frequencies between 0.015 However, it may not be wholly independent; nearly 5% of the and 0.05. SNPs assayed appear to be in some form of linkage disequilibrium with the mitochondrial lineages found in N. scabrosus, which suggests Population structure either a complex pattern of gene flow and introgression in the past, or 9 Global calculation of GST (0.025, P =0.001)andGST (0.046, P = a more complex interaction between mitochondrial and nuclear 0.001) using the 102 loci from BeadXPress arrays indicated statistically genomes as in some other incipient marine species (Willett and Burton significant population structure (see Figure 2). Eleven loci exhibited 2003; Burton and Barreto 2012). Interestingly, Hu (2008) showed 9 either GST or G ST greater than mean plus two standard deviations for that CND should limit the separation of neutral and selected clines, each statistic across all comparisons. However, the greatest values of and so the extent to which these genomes are linked makes the FST/GST are associated with small-sample comparisons with Niebla. pattern observed so far an unusual one. However, Hu (2008) also Results from STRUCTURE indicated that the most likely result notes that selective clines often will act as asymmetric barriers to the was for k = 2 populations, with individuals from Arica being spread of neutral alleles, a common result for introgression (Arnold statistically separate from individuals in central Chile. Analysis of 2006). Overall, this finding suggests that more information about molecular variance confirmed that approximately 3% of genetic Notochthamalus will be recovered from further exploration in loca- variance partitioned between regions defined by [1. Arica] and [2. tions to the south of Niebla; additional genotyping also will be u fi Las Cruces + Niebla], with RT =0.025(P = 0.01). The latter grouping necessary to increase the power of this test, as the ve loci indicated of individuals from Las Cruces and Niebla was used to test for CND, have only marginally significant results for the CND test; correction since both A and B mitochondrial lineages are found in these for multiple testing would, in fact, require a P-value of ,0.003, locations. which none of these loci obtain. Nevertheless, these results confirm the initial conclusions of Zakas Cytonuclear disequilibrium et al. (2009), which showed population genetic homogeneity in the fi Data from individuals in region 2 [Las Cruces and Niebla (n = 89 nEF1 gene region. Here we nd that although there is statistical sep- mitotyped with sufficient SNP data)] contributed to analysis of CND. aration of genetic data from a site in northern Chile (Arica) to one Of 102 loci, 5 (4.9%) exhibited significant (P , 0.05) CND either at that is over 2,000 km south (Las Cruces), the actual level of nuclear the allelic or genotypic level (Table 1). None of these loci contribute differentiation (GST of 0.025) is small relative to the ~2% sequence fi divergence and reciprocal monophyly between the two mitochondrial strongly to identi ed population structure, with mean GST =0.04and 9 lineages. The thousands of SNPs from our RADseq data support this G ST = 0.083. None could be reliably annotated using BLASTn or tBLASTx algorithms (results not shown). inference of nuclear homogeneity in central and northern Chile pop- ulations of N. scabrosus. Practically, these results also suggest that— for experimental and community ecology in the intertidal—there is DISCUSSION little reason to treat the two mitotypes as distinct species potentially in Exploring data that are more representative of whole genomes—and competition (Shinen and Navarrete 2010; Laughlin et al. 2012) across thus of the species—begins to provide more complete images of the the ~3000 km of coastline considered here. history of populations and the evolutionary mechanisms that are Our results from analysis of SNPs from RADseq data and direct associated with spatial patterns. Although the initial study of the genotyping via GoldenGate (BeadXPress) technology generated mitochondrial cline in N. scabrosus suggested that it was likely to have comparable patterns of allelic and genotypic diversity. These results originated via transient allopatry associated with the biogeographic compare favorably to those of Gautier et al. (2013b), which note that boundary at 30°S (Zakas et al. 2009), we now see that the nuclear pooled next-generation sequencing libraries generate comparable genome presents much less heterogeneity throughout the central and results as individual-based analyses, with lower cost and complexity. northern regions of Chile than the mitochondrial pattern would pre- Still, given concerns about the potential bias in estimates of genetic dict. Our attempt to analyze “pure” A- and B-mitotyped populations variation from RADseq data (Arnold et al. 2013; Gautier et al. 2013a), of individuals alongside geographic comparisons generated nearly it is important to weigh the potential for bias in these data prior to use identical—and quite low—patterns of population genetic variation. in larger-scale analysis.

228 | C. Zakas, K. Jones, and J. P. Wares The enigmatic discordance between the mitochondrial cline in Foll, M., and O. Gaggiotti, 2008 A genome-scan method to identify selected N. scabrosus, in which a highly divergent lineage appears at about 30°S loci appropriate for both dominant and codominant markers: a Bayesian and increases in frequency dramatically in populations to the south, perspective. Genetics 180: 977–993. and the nuclear genome—which exhibits relative homogeneity across Foster, B. A., and W. A. Newman, 1987 Chthamalid barnacles of Easter fi nearly 3000 km of coastline, is not a typical case of divergence in the Island; peripheral Paci c isolation of Notochthamalinae new subfamily and hembeli-group of Euraphiinae (Cirripedia: ). Bull. nuclear genome lagging behind that of the mitochondrial genome Mar. Sci. 41: 322–336. because of larger nuclear effective population size (Edwards and Gautier, M., K. Gharbi, T. Cezard, J. Foucaud, C. Kerdelhue et al., Bensch 2009). For a similar mitochondrial divergence pattern in the 2013a The effect of RAD allele dropout on the estimation of genetic confamilial barnacle Balanus glandula, spatially concordant nuclear variation within and between populations. Mol. Ecol. 22: 3165–3178. divergence is exhibited in multiple loci (Sotka et al. 2004; S. R. Palumbi, Gautier,M.,J.Foucaud,K.Gharbi,T.Cézard,M.Galanet al., 2013b Estimation personal communication). In B. glandula, both mitochondrial COI of population allele frequencies from next-generation sequencing data: pool and nEF1 loci exhibit a tight similarity in latitudinal transition from vs. individual-based genotyping. Mol. Ecol. 22: 3766–3769. northern to southern lineages. With similar data from a confamilial Hebert, P. D. N., E. H. Penton, J. M. Burns, D. H. Janzen, and W. 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Volume 4 February 2014 | Low SNP Variation in Notochthamalus | 229 biogeographic transition zone establish the occurrence of a sharp genetic Zakas, C., and J. P. Wares, 2012 Consequences of a poecilogonous life discontinuity in the kelp Lessonia nigrescens: vicariance or parapatry? history for genetic structure in coastal populations of the polychaete Mol. Phylogenet. Evol. 53: 679–693. Streblospio benedicti. Mol. Ecol. 21: 5447–5460. Toews, D. P. L., and A. Brelsford, 2012 The biogeography of mito- Zakas, C., J. Binford, S. A. Navarrete, and J. P. Wares, 2009 Restricted gene chondrial and nuclear discordance in . Mol. Ecol. 21: 3907– flow in Chilean barnacles reflects an oceanographic and biogeographic 3930. transition zone. Mar. Ecol. Prog. Ser. 394: 165–177. Wares, J. P., 2013 Mitochondrial evolution across lineages of the vampire Zakas, C., N. Schult, D. McHugh, K. L. Jones, and J. P. Wares, barnacle Notochthamalus scabrosus. Mitochondrial DNA. DOI: 10.3109/ 2012 Transcriptome analysis and SNP development can resolve popu- 19401736.2013.825791 lation differentiation of Streblospio benedicti, a developmentally dimor- Willett, C. S., and R. S. Burton, 2003 Environmental influences on epistatic phic marine annelid. PLoS ONE 7: e31613. interactions: viabilities of cytochrome c genotypes in interpopulation crosses. Evolution 57: 2286–2292. Communicating editor: K. Yuseob

230 | C. Zakas, K. Jones, and J. P. Wares